SUSPENSION CULTURE

Individual cells or cell aggregates dispersed and growing in moving liquid media is known as suspension culture.

Explants used in suspension culture
In regular practice suspension culture is initiated by transferring piece of undifferentiated and friable cells to a liquid medium which is continuously agitated by a rotator shaker.
Suspension cultures have also been started from sterile seedlings or imbibed embryos or leaves. Leaves and other tissues can be gently grinded using homogenizer. This homogenate containing living cells, dead cells and cell debris is cleared by filtration and centrifugation and then transferred to a liquid medium. This is the mechanical method
In enzymatic method, pectinases (enzymes which digest pectin cell wall) are used for isolation of single cells.
Method
At first stage culture are initiated by placing freshly cut sections of plant organs (root, stem, leaves) on a solidified nutrient medium
In this condition explants is transferred a liquid medium to obtain callus.
The callus is transferred to a liquid medium and agitated to obtain a fine suspension of cells.

Medium for suspension culture
A wide variety of media compositions have been used for suspension culture. These include MS, B5, LS (linsmaier and skoog) and Blaydes medium. For these media vitamins, inositol, glucose and growth regulators are incorporated.
Orbital shakers
In suspension cultures, orbital shakers are widely employed for initiation and serial propagation of plant cells. They should have a variable speed control (30-150rpm). They serve three main purposes.
They exert a mild pressure breaking the cell aggregates into single cells.
Agitation maintains uniform distribution of cells in the medium.
Movement of the medium provides good gaseous exchange.
Culture vessels
The Erlenmeyer flasks are commonly used as culture vessels. The volume of the culture medium should be appropriate to the size of the culture vessel:
Eg; 20ml/100ml flask
70ml/250ml flask
The flasks are normally sealed with aluminium foil.
Types of suspension culture:
1. Batch culture
2. Continuous culture
1. Batch culture:
The cell suspension culture grown in a fixed volume of nutrient medium is taken as batch culture. This is also known as closed system because the cells are incubated within a single batch of medium. The cells exhibit 5 phases of growth cycle. They are:
a. Lag phase
b. Log phase
c. Linear phase
d. Deceleration phase
e. Stationary phase

Lag phase:
Here the cells prepare to divide. It is the initial period pf the batch culture, where no cell division occurs, but the cells are metabolically active and the cell size increases, due to the synthesis of various components.
Log phase:
Here the rate of cell division is highest; here the cell division is exponential as the result there is an increase in cell number.
Linear phase
Here the cell division slows, but the rate of cell expansion (cell elongation) increase. After 3-4 generations, the cell growth declines.
Deceleration phase:
Here the rate of cell division and cell elongation decreases.
Stationary phase:
Here the number and size of cells remains constant. The doubling time in suspension culture varies for 24-48hrs. Cells should be sub cultured at weekly intervals.

2.Continuous culture:
Is one in which in flow of fresh medium is balanced by outflow of culture. In continuous culture the growth rate of cells and cell density are held constant by a fixed rate of addition of growth limiting nutrients and removal of cells and spent medium is; as there is increase in the cells, there is depletion of nutrients, therefore fresh medium is added and simultaneously equal volume of culture is removed. Hence this is also called as open system.



Continuous culture systems are of 2 types
1. Chemostat 2. Turbidostat

1. Chemostat
In this system growth rate and cell density are held constant by a fixed rate of a growth stimulating nutrient (nitrogen, phosphorous, glucose). In such a medium all constituents other than growth limiting nutrients other than growth limiting nutrient are present at a higher concentration than that is required.

2. Turbidostat
When there is an increase in turbidity of the culture, fresh medium is added and an equal volume of culture is removed.

Applications:
1. Production of secondary metabolites
2. Major source for obtaining individual cells which can be used for protoplast fusion and hybrid production.
3. Suspension cultures are used for induction of mutation and genetic manipulation of plant cells.
4. They can be preserved easily.